The analysis of samples by means of microscopy is a broad technical field for which a variety of technical solutions are available. Various microscopic methods have been developed on the basis of traditional light-optical microscopy,
Luminescence microscopy is a traditional field of application of light-optical microscopy for examining biological preparations. In so doing, certain dyes (also known as phosphors or fluorophores) are used for the specific marking of samples, e.g. of parts of cells. As mentioned, the sample is illuminated with excitation radiation and the luminescent light excited in this fashion detected with suitable detectors. A dichroic beam splitter combined with block filters is commonly provided in the light-optical microscope for this purpose, to separate the fluorescence radiation from the excitation radiation, thus allowing a separate observation. This approach makes it possible to reproduce individual, differently stained parts of cells in the light-optical microscope. Obviously, a plurality of parts of a preparation can be stained simultaneously with varying dyes that adhere specifically to different structures of the preparation. This method is known as multicolor luminescence. Furthermore, samples can be analyzed that are luminescent per se, i.e. without the addition of markers.
As is common, luminescence as used herein refers to the generic term for phosphorescence and fluorescence, meaning that it comprises both processes.
Moreover, the use of laser scanning microscopy (also known with the abbreviation LSM) has been disclosed. It only reproduces the plane located in the focal plane of a lens from a three-dimensionally illuminated image by means of a confocal detection arrangement (referred to as confocal LSM) or a nonlinear sample interaction (so-called multiphoton microscopy). An optical section is obtained and the documentation of a plurality of optical sections in various depths of the sample then makes it possible to generate a three-dimensional image of the sample by means of suitable data processing equipment, said image being composed of different optical sectional views. As a result, laser scanning microscopy is suitable for examining thick preparations.
Different approaches have recently been developed for resolutions beyond the diffraction limit, which is determined by the laws of physics. Said microscopic methods are characterized in that they provide the user with a higher lateral and/or axial optical resolution compared with a conventional microscope. In the description at hand, these kinds of microscopic methods are referred to as high-resolution microscopic methods, because they achieve a resolution beyond the optical diffraction limit. In contrast, microscopes with limited diffraction limits are referred to as conventional microscopes. They are used to realize known optical wide-field microscopy or laser scanning microscopy.
A high-resolution microscopic method is disclosed in EP 1157297 B1. In it, nonlinear processes are utilized by means of structural illumination. Fluorescence saturation serves as nonlinearity. The spectrum of lens space is displaced relative to the transmission function of the optical system through structured illumination. In detail, the displacement of the spectrum means that the lens space frequencies V0 are transmitted at a space frequency V0-Vm, wherein Vm is the structured illumination frequency. With the given maximum space frequency the system is able to transmit, this enables the transfer of space frequencies of the object that exceed the maximum frequency of the transmission function by the displacement frequency Vm. Fourier filtering as reconstruction algorithm for imaging and the use of a plurality of shots for one image are required for this approach. Structured wide-field illumination of the sample, for example through an amplitude/phase screen, is hence used in EP 1157297 B1, which is likewise fully taken into account with regard to the corresponding description of the resolution microscopy method. Fluorescence in the sample is again detected by wide-field microscopy. The screen is now brought into three different rotary positions, e.g. 0°, 120° and 240°, and in each rotary position, the screen is moved to three or more different positions. The sample is detected by wide-field microscopy for each of the three displacements of the three rotary positions (for a total of at least 9 illumination states). Furthermore, the screen has frequencies that are as close as possible to the limit frequency the used optical arrangement is capable of transmitting. The mentioned displacement subsequently takes place with the use of Fourier filtering, whereby in particular the zeroth and +/− first order of diffraction in the images is evaluated. Said microscopic method is also known as SIM method.
An increase in resolution is obtained with this principle if the intensity of the structuring (e.g. through a screen) is such that the fluorescence of the sample reaches saturation in the bright area. In that case, the structured illumination of the sample no longer has a sinusoidal distribution on the sample, but even higher order harmonics beyond the optical limit frequency due to the saturation effects. Said upgrade of the SIM method is also known as saturated patterned excitation microscopy (SPEM).
An upgrade of the SIM method can also be achieved with a linear illumination arranged perpendicular to the direction of the screen. This creates a line of illumination, whereby the screen structure is reflected along the line. In other words, the lines of the illumination are structured by the screen. The linear illumination allows a confocal slit detection and hence a further increase in resolution. Said method is also known as SLIM.
The publication by C. Muller and J. Enderlein titled “Image scanning microscopy”, Physical Review Letters, 104, 198101 (2010) is based on the SIM principle, although it scans the sample using confocal illumination and detection, followed by Fourier filtering. Said principle is also known as ISM. It does not involve nine orientations of a structured illumination, but each scan position, i.e. each scanning state during the scanning of an image corresponds to an illumination state, and the structured illumination is a spot illumination of the sample.
Another high-resolution method of luminescence microscopy is disclosed in the publication by T. Dertinger, et al., titled “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)”, PNAS (2009), p. 22287-22292 as well as “Achieving increased resolution and more pixels with Superresolution Optical Fluctuation Imaging (SOFI)”, Opt. Express, Aug. 30, 2010, 18 (18): 18875-85, doi: 10.1364/IE.18.018875 and S. Geissbuehler et al., “Comparison between SOFI and STORM”, Biomed. Opt. Express 2, 408-420 (2011). Said method is utilizing the blinking properties of a fluorophore. If the fluorophores of a sample are statistically blinking independently from each other, a considerable increase in resolution of the illustrated sample beyond the optical resolution limit defined by the laws of physics can be achieved by way of suitable filtering using a so-called cumulant-generating function. One example of said type of cumulant-generating function includes the second-order autocorrelation function. To generate a high-resolution image, it involves the recording of a series of individual images and the subsequent amalgamation into a single image by means of the cumulant-generating function, said single image then having the higher resolution. Said method is referred to as “Super-Resolution Optical Fluctuation Imaging” or SOFI method for short.
Based on the prior art, it is also known to combine a plurality of high-resolution microscopy methods. For example, the combination of a variety of high-resolution microscopy methods is described in DE 102008054317 A1, with the purpose of using the optimal method for individual sample areas, in each case taking into account the resolution and measuring speed.